Delta5-androstene-3beta, 17alpha-diol-16-one ethers



Patented June 29, 1954 A ANDROSTENE-3fl,17a-DIOL-16-lONE ETHEESvv Max N.Huffman, Oklahoma City, Okla., assignor to G. D. Searle & 00., Chicago,111., a corporation of Illinois No Drawing.- Application December 29,1951, Serial No. 264,247

This invention relates to compounds of thecyclopentanopolyhydrophenanthrene series and processes for manufacturingthe same. More particularly, it relates to the partial synthesis of the3,16,17-triol derivatives. of the estrogen and "androgen series ofcompounds and to the new intermediates which are produced in thesynthesis.

This application is a continuation-in-part of my copending applicationSerial No. 34,144, filed June 19, 1948, nowsPatent No. 2,584,271, datedFebruary 5, 1952. I

The products of this invention have certain physiological activity andthey may be used primarily to supplement the androgenic hormonesnaturally produced in the human body. Because of the possibility ofactivating various positions of the steroid nucleus and providingvarious functional groups on those activated positions, it isconceivable that new compounds unlike the natural occurring hormones maybe synthesized and used internally to impart physiological ac tivity ofa desired and predetermined character. Compounds of the type produced bymy invention may also be used as chemical intermediates for theproduction of other, cyclopentanopolyhw drophenanthrene derivatives.

It. is an object of this invention to effect the partial synthesis ofestriol, androstenetriol, and other 3,16,17-triols of the estrogen andandrogen series and to provide a method for producing the same.

Another object of this invention is to produce and to provide a methodfor manufacturing the a-kEtOlS of 3,17-dio1 derivatives of the estrogenand androgen series of compounds including the corresponding 3-alkylethers, such as A -androstene-3 (,3)',17(a) -diol-16"-one-3-alkyl etherin which the alkyl group may be methyl, ethyl, propyl, benzyl, and thelike; the 16,1-7'-dihydr.oxy derivatives of the estrogen and androgenseries of compounds including estriol, isoestriol-A, the Hirschmanntriol, and the like; the derivatives of 16,17-dihydroxy estrogen andandrogen series of compounds including the cyclic acetal ofisoestriol-A, isoestriol acetonides; and the alkyl ethers of. the16,17-dihydroxy estrogen and androgen series of compounds, such 'as A-androstem-3(5) ,16( 3) ,17 (a) -triol-3-alky1 ethers including [1-53(5) ,16 (p) ,1701) -triol-3-methyl ether 3 Claims. (Cl. 260397.4)

and other derivative ethers in which aliphatic, aromatic, and mixedaliphatic-aromatic groups are substituted for, methyl.

A further object is to provide a method for converting 16-oximino.steroid derivatives, such as compounds of the estrogen and androgenseries having. a functional oxygen containing group on the C3 and Cupositions, to the corresponding a-ketols (vicinal ketols) or16,17-ketols by a reduction process; converting the estrogen andandrogen series of compounds having at least one ketone group in the 16and 17 positions and a hydroxy group inv the other if only one of the lv positions is ketone to the corresponding 16,17-

dihydroxy derivatives by a reduction process which does not give rise inlarge proportions to a 16,17 configurationv differing from that of thenaturaloccurring estriol; separating the cisoid and transoid16",17-dihydroxy derivatives of the estrogen and androgen series ofcompounds; and

the dealkylation of the 16,17-dihydroxy-3-alkyl ethers of the estrogenand androgen series of compounds and the conversion thereof to thecorresponding triols.

By the term "cyclopentanopolyhydrophenanthrene compounds, as usedherein, it is meant the group of substances based upon the followingnucleus,

in which R may be hydrogen, the rings A, B, C, and D may either besaturated on unsaturated and in which some of the rings, such as rings Aand B may have as much as three unsaturated groups to comprise benzenoidstructures; Included among the compounds are the steroids of theestrogen and androgen series which find ene), equilenin (3 hydroxy 17keto A estrapentaene), equilin (3 hydroxy 17 keto A -estratetraene), andandrogen compounds 'androsterone (3(a) hydroxy 1'7 keto andro-Representative of such replaceethyl, propyl, and the like aliphatics,benzyl,

naphthyl, and the like aromatics or mixtures thereof and derivativesthereof., Although my invention may be practiced by starting with one ofthe original materials described, inventive features of my inventioncommence by the reaction with the 16-o'ximino derivative of thecorrespond-* ing steroid. t In the steps by which one of the compoundsnamed above, such as estrone, is converted to the corresponding16-oximino derivative, the 3-hydroxy group may be etherified, that is,methylated by the process described by Butenandt, Stormer, and Westphal,Z. Physiol. Chem., 208 (1932). The corresponding 3-alkyl ether may thenbe nitrosated to give the 16-oximino derivative by the process of Litvanand Robinson, Journal of the Chemical Society, page 1997 (1938).

In carrying out my invention, the 16-oximino derivative of thecorresponding steroid is carried through a reduction reaction by whichthe vicinal a-kBtOl derivative is produced. The oc-kGtOl in crude orpurified form is then subjected to another reduction reaction to producethe a-glycol derivative hereinafter referred to as the corresponding16,17-dihydroxy steroid. This includes 16,17 -dihydroxy steroids inwhich the 3 position is either hydroxy or otherwise modified, such as byan alkyl ether group. This last reduction step ordinarily results in theproduction of two isomeric glycols; namely, the transoid 16(5) ,1'7(a)-dihydroxy steroid and the cisoid 16(oc),17(o -dihydroxy steroid. Ifdesired, these may then be separated. I have conceived two processes bywhich these isomers may be separated. One includes a fractionalcrystallization process from solvent solution. The other requires areaction of the isomeric mixture with a substance which forms thederivative of only one enabling it to be separated from the other bysuch means as selective solvency. Each of the compounds resulting fromthe latter process may be reconverted to their original 16,17-dihydroxyform. When the active C3 position has been protected by an ether group,the e-glycol may be dealkylated to produce the corresponding triol.

The 16-oximino or isonitroso steroid may be reduced to the correspondinge-ketol by the Stodola reduction, Stodola, Kendall, and Mc- Kenzie,Journal of Organic Chemistry, 6, 841 (1941). Briefly described, theprocess includes the reaction of the lfi-oximino steroid derivative withzinc dust in the presence of an organic acid, such as acetic acid, whichmay or may not be diluted with water. Upon evaporation of the separatedliquid phase, a crystalline u-ketol is produced which may be used as is,such as in the subsequent production of the corresponding triol, or theproduct may be purified by extraction with 4 solvent, such as an etherin dilute alkali solution. The ether phase may be washed by one or moreof the compositions including dilute mineral acids, such as hydrochloricacid, dilute alkali metal bicarbonates, such as sodium bicarbonate, orwater.

It is by this method that I have succeeded in producing compounds suchas A -androstene- 3(fl),17(a)-dl01-16-0I18 and A- -androstene-3 (5)'17(1)-diol-16-one-3-alkyl ethers, such as the B-methyl ether, 3-ethylether, 3-benzyl ether, and. the like. To the best of my knowledge, thesealkyl ethers have not heretofore been produced by natural glandularsecretion or by synthetic processes.

A salient feature of this invention resides in the use of a metalamalgam to efiect the reduction of the c-ketol to the corresponding16,1'7-dihydrcxy steroid which may be a trial if C is hydroxyl or aglycol if the C3 position is otherwise modified, such as ,by an alkylether. When the reduction from the uketol is carried out by hydrogen inthe presence of a catalyst, the product is undesirable because itconsists essentially of isomers of a 16,17 configuration which differsvfrom that of the natural occurring triols, such as estriol. Excellentyieldof 16,17-dihydroxy steroids of the desired characteris secured whenthe reduction is carried out with a metal amalgam, preferably in thepresence of a. hydrogen donor and at a temperature below 50 0. As themetal amalgam, I make use of mercury in combination with one of the basemetals, such as sodium, zinc, magnesium, and the'like. In the reaction,an excess of the metal amalgam at relatively. low concentration, such asabout 2 per cent, is used. Suitable hydrogen donors for use in the abovereaction include inorganic or organic acids, bases, and neutralmolecules of the type water and alcohol. As the hydrogen donor, I preferto use a substance in which the steroid derivative is soluble. One suchcomposition from which I have obtained excellent yield consistsessentially or alcoholic acetic acid and freshly prepared sodium amalgamof about 2 per cent concentration. Excellent yields of the desiredglycols are secured when the reduction reaction is carried out at atemperature of about 40-41 C. or less. When higher temperatures areused, and especially when temperatures in excess of 50 C. are used,lower yields of the desired transoid derivative are secured.

Refinement of the l6,l7-dihydroxy steroid resulting from the reductionprocess may be effected by extracting the reaction product with waterand a suitable water immiscible solvent in which the steroid derivativeis soluble, such as most of the higher molecular weight liquid a1cohols, including butanol and the like, esters, such as ethyl acetate,amyl acetate, and the like, and ethers, such as ethyl ether, propylether, etc. A suitable solvent system, for example, comprises water andethyl ether. The separated ethereal phase may be subsequently extractedwith water and/or dilute alkali solutions, such as a sodium hydroxidesolution, before evaporation of the ether to crystallize the resultant16,1'7-dihydroxy steroid. derivatives.

Another feature of this invention resides in the novel methods by whichthe two isomers of 16,1'7-dihydroxy steroids, that is, the cisoid andtransoid isomeric substances, may be separated one from another. Amixture of these isomers is usually produced by the described metalamalgam reduction. One isomer which is produced in greater proportionwhen the reduction reaction temperature is held below 50 C. is thetransoid 16(5) ,17'( u)-dihydroxysteroid. The other less desirable isthe cisoid 16(w)-,1'7( 1) -dihydroxy steroid.

-I have foundthat the transoid may be rather completely separated fromthe cisoid form by the process of fractional. crystallization fromsolvent solution, especially when the 3 position isalkylated. Because:the transoid is less soluble than'the cisoid in. non-polar or relativelynonpolar solvents, itmay be separated from the cisoid by one or morerecrystallizations from non-polar solvent systems. drocarbonsg. such asthe petroleum distillates, coal tar distillates, includingpetroleumethers, cyclohexane, toluene, benzene, butane, and otheraliphatic and alicyclic solvents, and the like are examples of non-polarsolvents. Ordinarily, an organic liquid compatible with the non-polarsolvent and in which the steroid derivative is soluble is incorporatedinto the solvent system to' control the degree of solvation. I may makeuse of alcohols, ketones, ethers, dioxanes, and the like for the lattercomponent. I prefer to use a solvent component which has a lowerboiling-point or greater volatility than the nonpolar' constituent ofthe solvent system to enable partial removal thereof from the solventsystem When' desired. One suitable solvent, system with which I haveobtained excellent results makes use of acetone and cyclohexane.

When the 3 position is unalkylated, or phenolic, the transoid form maybe separated, by another method, from the cisoid form by subjecting themixture to an acetal forming medium to. form the cyclic acetal of thecorresponding cisoid 16(oc) ,l7(oc) -dihydroxy steroid. The transoidderivative which is substantially unreacted by the acetal forming mediummay then be separated from the cisoid cyclic acetal by ordinaryselective solvent means. A single solvent system may be used in whichthe tolerances for the cisoid acetal and the transoidglycol are of adifferent order enabling separation by fractional crystallization. A twocomponent solvent system may be employed in which the selective solventprinciple operates to concentrate one derivative in one phase and theother derivative in the remaining phase which is separable from thefirst. The latter system may include an aqueous alkaline phase in whichthe transoid derivative concentrates and an immiscible organic solventphase for the cisoid cyclic acetal, but in this event, it is. desirablethat the 3' position be phenolic. For example, when the latter system isused, thetransoid form is found in high concentration in the solventcomponent containing an aqueous base solution, such as a dilute sodiumhydroxide, potassium hydroxide solution; and the like. The alkalinephase, which is separable from the solvent phase, may be extracted witha solvent for the cyclic acetal to extract anytraces thereof. Theaqueous. hydroxide phase may then be acidified to produce thecorresponding triol.. The solvent phase may be a solvent immiscible withthe aqueous alkaline solution and in which the cisoid cyclic acetal issoluble. Representative of such solvents are the chlorinated solvents,ethers, ketones, and some of the aromatics. Chloroform, ether, ethylacetate, and ethylene dichloride are specific examples. The glycol maybe regenerated by hydrolyzation of the cylic acetal.

As. the acetal forming medium, ketones and aldehydes capable of reactionin the presence of a catalyst, suchas an acid, to efiect. the acetal Theordinary hy-.

reaction may be used. -Acetone, methyl ethyl ketone, and other ketonic'solvents or other ketones represent suitable acetal forming compounds;aldehydes, such as butyraldehyde, glyoxal, pyruvic aldehyde, andaldehydic solvents, such as benzaldehyde, may be used: Inorganic acids,represented by anhydrous hydrochloric acid or otherhalogenic acids,comprise catalysts for the acetal reaction. I have secured excellentresults by the use. of an acetal forming system consisting of acetone,only a small portion of which is first saturated with anhydrous hydrogenchloride.

It-is by' the described method that I have succeeded for the first timein producing the cyclic acetal of isoestriol-A, such asisoestriol-A-acetoni'de; and other isotriol-A-acetonides of the estrogenand androgen series of compounds.

It is by these methods that I have succeeded for the first time inpreparing and isolating the 3'-alkyl ethers of A -androstene-3(c),l6(;8) ,17 (a) triol including the 3-methyl ether, the 3-ethyl ether,the S-benzyl ether, and the like.

As previously pointed out, the e-glycols, such as the reaction productsof the metal amalgam reduction, may be constituted with an alkoxylgroup, such as methoxyl, at C3. When desired, conversion to thecorresponding triol may beeffected by a simple d-e-etherification,v suchas. by hydrolysis. When the C3 position may be either (a) or (B), as inthe androstane" series of compounds, demethylation or dealkylationpresents a very difficult problem.

I have discovered a very novel reaction by which the methylgroup may beeasily and smoothly replaced in quantitative yield by a group readilyconvertible to hydroxyl. I first form the diacetate by reaction of theglycol with acetic acid or acet c anhydride; then I react the diacetatewith p-toluenesulfonic acid in the presence of acetic anhydride tosubstitute acetyl for the methyl group at C3. The resulting demethylatedsteroid may be saponified insolvent solution by an: alkali. metal baseand from which the triol may be regenerated by simple hydrolysis in acidmedium.

By way of illustration but not by way of limitation, the followingvexamples are given to illustrate the invention.

EXAMPLE 1 The conversion of estrone (I) to estronemethyl ether (II Themethylation of estrone may be carried out by the process of Butenandt,Stormer, and Westph'al as previously pointed out.

EXAMPLE 2 The conversion of estrone methyl ether ('1!) to16-0zrimiho'estrone-d-methyl ether (III) This. 'nitrosation reaction maybe carried out according. to the process described by Litvan andRobinson, Journal Of The Chemical Society, page 1997 (1938).

EXAMPLE 3 The reduction of 16-oximino-estrovte-3-me1zhyl ether (HI)- to16-keto-1-7-hydromy estmtrienbl- S-methyl ether (I V) maintained at 45C. until solution of the steroid derivative was complete. Then 9.4 cc.of water were added and the solution refluxed for 1 hour 10 minutes andcooled to room temperature. The aqueous acetic acid solution wasdecanted from the zinc, and the latter washed with 100 cc. of benzene.To the combined aqueous acetic acidbenzene solutions were added 80 cc.of 1.5 N sodium hydroxide and 200 cc. of ethyl ether. After having beenpartitioned and separated, the organic phase was washed successivelywith 0.5 N hydrochloric acid, with 5 per cent sodium bicarbonate, andwith water. Evaporation of the ethereal solution yielded a crystallineproduct which, after treatment with charcoal, was recrystallized oncefrom cyclohexane and once from aqueous ethanol to give 207 mg. ofneedles melting at 164-165 C. Another treatment with charcoal andrecrystallization from aqueous acetone raised the melting point to167-168 C. (203 mg.). The latter purification step may be dispensed within the event that the crude reduction product is siutable for use or forfurther reaction as desired.

EXAMPLE 4 Reduction of 16ktO-17(oz) -hydro:1:y estratriemol- 3-methylether (IV) to 16,17-dz'hydroxy estratrienoZ-3-methyl ether (V) Asolution of 800 mg. of the a-ketol methyl ether in 100 cc. of ethano1and 10 cc. of acetic acid was carefully maintained at 40 C. (waterbath), and 200 gm. of freshly prepared sodium amalgam (2 per cent) wereadded in small pieces with efficient swirling. Before all of the amalgamhad been added, a precipitation of sodium acetate occurred, and at thispoint an additional 10 cc. of 50 per cent acetic acid were added. Afterall the reducing agent had been added, the mixture was transferred to aseparatory funnel with ether and water. The mercury plus aqueous phasewas separated, after partitioning, from the ether; the latter may befurther washed with water, with 0.5 N sodium hydroxide, and again withwater to purify the m-glycol. Evaporation of the ethereal phase yieldeda crystalline residue of the isomeric transoid16(}3),1'7(0z)dlhydroxy-steroid-3-methyl ether and cisoid 16(c), 17 (a)-dihydroxy-steroid-3-methy1 ether.

EXAMPLE 5 Separation of the cisoid from the transoid 16,17

glycols of steroid-.l-methyl ether (V) by I tional crystallizationSeparation was effected by fractional crystallization from non-polarsolvents system in which the transoid derivative was less soluble thanthe cisoid. To this end, the mixed a-glycols were recrystallized twicefrom acetone-petroleum ether. In actual practice, I dissolve the mixtureof isomers in acetone and then add about 2 volumes of the non-polarpetroleum ether. Sinc the acetone has a greater volatility than thepetroleum ether, the former leaves the solution and at the point wherecrystallization is initiated, further evaporation is stopped andcrystallization, such as by cooling, causes the desired separation.

EXAMPLE 6 Demethylation of estrioZ-3-methyl ether (V) to produce estriol(VIII) To a solution of 155 mg. of estriol-3-methyl ether in 3 cc. ofpyridine were added 3 cc. of acetic anhydride; the phases were mixedwell and left 48 hours at room temperature. The diacetate was thenprecipitated at the addition of cold water, and, after having beenallowed to stand several hours, filtered, washed well with water, anddried in vacuo.

The dry diacetate was then dissolved in 5 cc. of warm acetic acid, 5 cc.of freshly distilled hydriodic acid (specific gravity 1.7) were added,and the solution of diacetate methyl ether refluxed at once over a freeflame. After exactly 5 minutes refluxing cc. of cold aqueous sodiumbisulfite were added and the resulting phases mixed well. Afterprecipitation had become complete (ice box), the crystalline steriod wasfiltered, washed with aqueous sodium bisulfite, and then washed wellwith water. The residue was dried in vacuo.

The demethylated estrogen was then dissolved in 25 cc. of ethanol (roomtemperature) and a small spatula end of sodium hydrosulfite added; Tothe alcoholic solution were next added, for purpose of saponification,25 cc. of 1 N sodium hydroxide, and the latter was mixed with rapidswirling. The resultin solution at first became colored a deep yellowbut soon turned almost colorless. After 24 hours saponifications at roomtemperature, the ethanolic alkali was added to 250 cc. of 5 per centsodium bisulfi-te containing 2.1 cc. of concentrated hydrochloric acid,and the resulting voluminous precipitate extracted with 500 cc. of ethylether. The ether, after having been partitioned and separated from theaqueous phase, was washed once with 250 cc. of 5 per cent sodiumbisulfite, twice with 200 cc. portions of 0.5 N sodium hydroxide. Thecombined sodium hydroxide phases (400 cc. of 0.5 N) were acidified withconcentrated hydrochloric acid and extracted with 500 cc. of ethylether. This ether, after separation from the aqueous acid phase, may befurther washed with bicarbohate and with water; a crystalline residue ofestriol resulted on its evaporation. The crystalline residue (aftertreatment with charcoal) may be recrystallized once from aqueous ethanolto give estriol.

EXAMPLE 7 Reduction of 16-oximinodehydro[isoandrosterone] 3-methyl ether(IX) to M-[androstenel- 3(3),17(a)-diol-16-oaze-3-methyl ether (X) To500 mg. of 16-oximinodehydroisoandro sterone-3-methyl ether were added34 cc. of 50 per cent acetic acid and 1.4 gm. of zinc dust. The mixturewas refluxed for 1 hour and the hot solution decanted from the zinc into175 cc. of water. The zinc was rinsed twice with 4 cc portions of aceticacid and these united with the main reaction solution in water which wassubsequently partitioned with 200 cc. of ethyl ether; the latter, afterseparation, was washed with 0.5 N hydrochloric acid, with 1.0 N sodiumhydroxide, and with water. Evaporation of the ethereal phase yielded awhite crystalline product, A -androstene-3 (B) ,17(oc) -diol16-one-3-methy1 ether.

EXAMPLE 8 Reduction of A -androstene-3(fi),17(a) -dioZ-16- one-3-methylether (X) to A -andros-tene- 3(,8),16(B),17(a)-triol-3-methyl ether (XI)by sodium amalgam To the solution of the a-ketol, of Example 7, in 58cc. of ethanol and 6.5 cc. of acetic acid were gradually added gm. of 2per cent sodium amalgam, the temperature being carefully maintained at40.0-40.5 C. by continuous swirling in a water bath. (As soon as sodiumacetate comacsasnls 9 menced-to precipitate, 5.0 cc.of :50 per centacetic acid were added.) When the reaction had been completed, .themixture of mercury and solution was diluted with water and extractedwith ethyl ether. After removal of the mercury .and aqueous phases, theether may be further washed with water, with 0.5 N sodium hydroxide,:andagain with water, Evaporation of the ether yielded a whitecrystallineproduct. )After recrystallization, twice fromacetone-petroleum ether and once from absolute acetone A' -androstene-318) ,16(fi) 17(u)-trio1-3-methyl ether crystallized as .the hemihydratein the form of tiny leaves. The cisoid .derivative is to be foundinthepetroleum solvent.

EXAMPLE 9 Transformation of urinary androstene'triol-3- methyl ether(XI) to the triol (XII) To 301 mg. of the 3.::1ethyl ether .of Hirsch-'-manns triol (M. P.'195-196 C.) dissolved in 5 cc. of dry pyridine wereadded 3 cc. of acetic anhydride. Thetwo phases were mixed well and left24 hours with occasional swirlingi The diacetate was then precipitatedby treatment-with 200 cc. of ice water, and finally, after 3 hoursin theice box, filtered and washed copiously with water.

To the air-dried diacetate "(as above) were added 186 mg. ofp-toluene-sulfonic. acid monohydrate and the solid materials coveredwith 18.6 cc. of redistilledaceti'canhydride. The mixture was heated onthe steam bath with frequent stirring for exactly 30 minutes and thencooled in an ice bath. As no crystallization of triace'tate had occurredwithin 3 hours time, the anhydride was decomposed with ice water andthestero'id extracted with'ethyl ether. Theether, afterhaving beenwashed with aqueous sodiumbicarbomate and with water, was dried and thenevapo rated to produce the triacetate. The resulting white residue maybe recrystallized from aqueous ethanol (one time with theaid ofcharcoal) to yield 249 mg. of I white plates, P. 183-18 5''=C.

All filtrates from the recrystallizations of triol triacetate were addedto ether and the ethereal solution washed several times with water.After evaporation of this ether, the resulting crystalline residue wasdissolved in 25 cc. of ethanol, 25 cc. of 1.0 N potassium hydroxide werethen added, and the alkaline solution refluxed for 30 -minutes.Following the addition of 25 cc. of water, the saponification medium wasdistilled until it had become turbids The free triol was allowed tocrystallize for 2 days in the ice box and then filtered and washed wellwith water (100 mg, melting at 250252 C.). This material may berecrystallized once from aqueous ethanol, once from absolute acetone,and again from aqueous ethanol to produce the triol crystallized ashexagonal plates with one-half molecule of water.

EXAMPLE The sodium amalgam reduction of 16-kZtO-ozestmdz'o l (XIII) toestriol (VIII) To a solution of 0.44 g. of 16keto-u-estradiol in 50 cc.of 95 per cent ethanol plus 5 cc. of acetic acid were gradually added100 g. of 2 per cent sodium amalgam, with the application of continuousand efiicient mixing. The temperature of the reaction bath was carefullymaintained at 40.040.5 C. As soonas sodium acetate commenced toprecipitate from the reaction mixture, 5 cc. of 50 per cent acetic acidwas added. The supernatant fluid was decanted from the EXAMPLE 11 Theseparatiba-bfthe cisoid and transom isomers of estriol Thenon-ketonicproduct of Example 10 was dissolved in 35 cc. of acetone atroom temperature and 5 cc. of acetone saturated with anhydrous hydrogenchloride was added. This acetal forming medium was swirled for 5 minutesor longer-and then added to 50 cc. of aqueous potassium carbonate(containing 1.50 g. of KzCOs), mixed ationce', and then further dilutedto a volume of 400 cc. with water. After a day in the ice box, theprecipated material was filtered, washed with water containing a traceof pyridine, and then dried in ,vacuo. The dried residue, containing thecisoid form as the acetal, was then dissolved in 300 cc. of chloroformand partitioned with 300 cc. of 0.1 Nsodium hydroxide. The alkalinephase, containing the transoid form as the alkali metal hydrate, wasseparated andvagain washed withe300 cc. of chloroform. The sodiumhydroxide phase was then acidified with hydrochloric acid and extractedwith ethyl ether. Evaporation-of the ethereal phase yielded estriol,which may be recrystallized to :any stage of purity desired. .From thecombined chloroform washings containing the cyclic acetal of the cisoidderivative may be obtained isoestriol- A acetonide (XIV) (M. P.183.5-184.5 C.). Mild hydrolysis of isoestriol-A acetonide gives pureisoestriol-A.

EXAMPLE 12 The reduction 07 IG-ICGtO-oc-GSiTddiOl (XIII) to estriol bymagnesium amalgam To a solution of 485 mg. of ld-keto-a-estradiol in 50cc. of ethanol cooled in an ice-water bath was added 76 g. of magnesiumamalgam (4 g. Mgz72 g. Hg), then gradually over the course of 1 hour wasadded 20 cc. of aqueous acetic acid (45 parts HOAc:13.5 parts H2O) in 1(:0. portions. Temperature was not allowed to exceed 50 C. After thereaction was ended, the mixture was transferred to a separatory funnelusing 800 cc. dilute hydrochloric acid and 800 cc. of ethyl ether. Afterhaving been partitioned, the ether was separated and washed again withdilute hydrochloric acid and with water. Evaporation of the etheryielded estriol. This may be further treated by the processes ofExamples 11 or 5 to separate estriol from isoestriol-A.

The reactions may be illustrated by the'following formulae which showthe various transformations described.

It will be understood that many changes and variations in materials,concentrations, and conditions may be made by those skilled in the artin accordance with the principles set forth without departing from thespirit of the invention as claimed.

The stereochemical designations at positions 16 and 17 employed in thisapplication and in the parent application Serial No. 34,144, filed June19, 1948, now Patent No. 2,584,271, dated ll 12 February 5, 1952,conform to the terminology GE GE used in Gilman, Organic Chemistry,second 03 edition, 1943, volume II, chapter 19, John Wiley OH: A 0H; andSons, Inc., New York, N. Y., and by Hufi- FN OH 0 man and Lott, J. Am.Chem. 800., '71, 719 (1949). 5

CH: CH:

CH 0 CH 0 xx x CH3 OH;

OH OH HO cmo OH OH OH: cm

. 0H O omo Ho CH: OH:

0H 0 n omo omo o 0/ \B CH: CH:

OH- H0 011 OH x111 XIV I claim:

1. A -androstene-3(p), 170x) dio1-16-one-3- lower alkyl ether.

011.0 HO 2. M-androstene-Mp), 17(u) diol-16-one-3- methyl ether.

v VIII 3. A -androstene-3( 8), 17(a) dio1-16-one-3- benzyl ether.

References Cited in the file of this patent Huffman et aL, Jour. Biol.Chem. 172, 789-795 (1948).

1. $5-ANDROSTENE-3(B), 17(A) - DIOL-16-ONE-3LOWER ALKYL ETHER.